A problem encountered in the practice of medicine when using proteins as injectable pharmaceuticals is the frequency at which those injections must be made in order to maintain a therapeutic level of the protein in the circulation. For example, erythropoietin has a relatively short plasma half-life (Spivak, J. L., and Hogans, B. B., Blood, 73:90 (1989); McMahon, F. G., et al., Blood, 76:1718(1990)). Therefore, therapeutic plasma levels are rapidly decreased, and repeated intravenous administrations must be made. An alternative route of administration is subcutaneous injection. This route offers slower absorption from the site of administration, thus causing a sustained release effect. However, significantly lower plasma levels are achieved and, thus, a similar frequency of injection, as is required with intravenous administration, must be used to produce a comparable therapeutic effect.
Modification of naturally occurring proteins which have therapeutic value is often attempted in an effort to increase the protein's biological activity. Several methods have been employed to increase the biological activity of therapeutic proteins. These methods often focus on increasing the size of the therapeutic agents. For example, the size of a protein can be increased through chemical conjugation with a reagent such as polyethylene glycol (PEG) (Knusli, C. et al., Brit. J. Haematol, 82:654-663 (1992)). This procedure, also known as "PEGylation", has been reported with several protein agents, first as a means to reduce antigenicity, but also as a way to increase biological activity.
Another method of increasing a protein's size is through chemical cross-linking with another protein. For example, to increase the antigenicity of a protein, chemical cross-linking agents are used to conjugate the immunogenic protein to a carrier molecule such as immunoglobulin or serum albumin.
However, the conjugation of chemical compounds or inert molecules to a protein often results in a significant decrease of the overall biological activity, and of selected biological activity of the protein. (Knusli, C., et al., Brit. J. Haematol., 82:654-663 (1992)). These conjugations must be designed such that the resulting modified protein remains therapeutically efficacious and retains the desired biological properties of the unmodified, wild type (i.e., naturally-occurring) protein (Satake, R., et al., Biochem. Biophys. Acta. 1038:125-129 (1990)). Thus, it would be advantageous to be able to modify therapeutically active proteins to increase their biological activity which would result in less frequent injections or smaller doses of protein.